For storage stability and convenience of handling, proteinaceous pharmaceutical compositions are often formulated as a lyophilized (i.e. freeze dried) or vacuum dried powder and stored at low temperatures between −10 C to 4 C. The dry powder is reconstituted with a suitable fluid, such as saline or water, prior to administration to a patient. Alternatively, a proteinaceous pharmaceutical composition can be formulated as an aqueous solution or suspension that includes a stabilizer to prevent protein degradation. However, many proteins are very difficult to stabilize resulting therefore in a loss of protein and/or protein activity during the formulation, reconstitution and/or the period of storage. Stability problems can occur as a result of protein denaturation, degradation, dimerization, and/or polymerization. Various excipients, such as albumin and gelatin have been used with differing degrees of success to try and stabilize a protein active ingredient in a pharmaceutical composition. Cryoprotectants such as alcohols have been used to reduce protein denaturation under the freezing conditions of lyophilization. Stabilizers can work by reducing adhesion of the protein active ingredient to surfaces, such as the surfaces of laboratory glassware, vessels, the vial in which the pharmaceutical composition is reconstituted or the inside surface of a syringe used to inject the pharmaceutical composition. Adhesion of a protein active ingredient to surfaces leads to loss of active ingredient and can also contribute to denaturation of the remaining retained protein active ingredient, both of which reduce the total activity of the active ingredient present in the pharmaceutical composition. Stabilizers can also reduce denaturation of the active ingredient which can occur upon preparation of a low dilution solution of the active ingredient. As well as being able to stabilize a protein in a composition, an ideal stabilizing agent should have negligible immunogenicity when injected into a human patient.
Pure botulinum toxin is so labile that it is has limited practical utility to prepare a pharmaceutical composition. Botulinum toxin complexes, such as the toxin type A complex are extremely susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. Inactivated toxin forms toxoid proteins which may be immunogenic. The resulting antibodies can render a patient refractory to toxin injection. As with enzymes generally, the biological activities of the botulinum toxins is dependent, at least in part, upon their three-dimensional conformation. Additionally, it is known that dilution of the toxin complex obtained by the known culturing, fermentation and purification to the much lower toxin concentrations used for pharmaceutical composition formulation results in rapid detoxification of the toxin unless a suitable stabilizing agent is present. Dilution of the toxin from milligram quantities to a solution containing nanograms per milliliter presents significant difficulties because of the rapid loss of specific toxicity upon such great dilution. Current stabilizers that have been used in botulinum toxin formulations are animal derived albumin and gelatin. However, these stabilizers are not able to sufficiently stabilize botulinum toxin for storage at room temperature. Thus there is a specific need for enhancement of the stability of botulinum toxin and also to promote its permeation through the skin.
The process that leads to skin aging and wrinkles is complex. A primary cause of wrinkling is a build-up of free radical toxic plaque that binds to collagen and elastin fibers, causing the skin's supportive structure to become inflexible and unhealthy. Laugh lines, smile lines, crow's feet or facial creases appear in areas where repeated muscle movement occurs.
In 2002 the FDA approved the Botulinum Toxin Type A (BOTOX® Cosmetic) to temporarily improve the appearance of moderate to severe frown lines between the eyebrows (glabellar lines). BOTOX® Cosmetic is a purified protein produced by the Clostridium botulinum bacterium. The toxin reduces the activity of the muscles that cause frown lines between the brows to form over time. While BOTOX® Cosmetic has been successful in some cases, there are areas where it should not be used, such as throat and neck areas, around the mouth, near the eyes, and the hands. If the muscles in these areas are paralyzed or disabled, it can lead to difficulty talking, smiling, eating, swallowing, moving hands and fingers, and seeing.
Current formulations of BOTOX® Cosmetic must be administered within four hours after reconstitution since the toxin molecule is very labile. During this time period, reconstituted BOTOX® Cosmetic is stored in a refrigerator (4.degree. C.). Breakdown of the toxin into toxoid can induce immune responses to the toxoid that can interfere with subsequent treatments.
Animal derived or donor pool proteins such as gelatin and serum albumin have been used with some success to stabilize botulinum toxin. However, there has been a desire for improved methods of stabilizing botulinum toxins.
It has been suggested that a suitable alternative to human serum albumin as a botulinum toxin stabilizer may be another protein or a low molecular weight (non-protein) compound (Carpender et al., Interactions of Stabilizing Additives with Proteins During Freeze-Thawing and Freeze-Drying, International Symposium on Biological Product Freeze-Drying and Formulation, Oct. 24-26 1990; Karger (1992), 225-239. However, many substances commonly used as carriers and bulking agents in pharmaceutical compositions have proven to be unsuitable as albumin replacements in compositions containing Clostridial toxin. For example, the disaccharide cellobiose has been found to be unsuitable as a botulinum toxin stabilizer. The use of cellobiose as an excipient in conjunction with albumin and sodium chloride was reported to result in a much lower level of toxicity/efficacy (10% recovery) after lyophilization of crystalline botulinum toxin type A with these excipients, as compared to the toxicity after lyophilization with only human serum albumin (>75% to >90% recovery). Goodnough et al., Stabilization of Botulinum Toxin Type A During Lyophilization, App & Envir. Micro. 58 (10) 3426-3428 (1992). Schmidt, et al., Endoproteinase Activity of Type A Botulinum Neurotoxin Substrate Requirements and Activation by Serum Albumin, J. of Protein Chemistry, 16 (1), 19-26 (1997).
Gelatin has been used in some protein active ingredient pharmaceutical compositions as an albumin substitute. However, it does not provide for stabilization of botulinum toxin at room temperature.
Several efforts have been made to provide botulinum toxin formulations that can be stabilized and delivered in alternative ways. For example, U.S. Pat. No. 6,585,993 discloses a biocompatible implant for continuous release of a neurotoxin over a treatment period extending from one month to five years. While such an implantable system may be useful for certain situations, such as for the treatment of migraine, this type of implant system is not feasible for the treatment of facial, neck or hand wrinkles.
United States Patent Application No. 2004/0247623 suggests a method for the treatment of sensory neuron related distorters through transdermal application of a neurotoxin. This application is particularly directed to a method of treating migraine. The application suggests that botulinum toxin can be administered transdermally through a variety of ways. For example, the toxin may be incorporated into a transdermal patch or it may be administered through electrophoresis. The application also suggests that botulinum toxin can be administered using a topical cream. They teach that this would be achieved by reconstituting botulinum toxin with normal saline and then mixing the reconstituted toxin with a suitable cream or base and then massaging it on to the affected area. This type of application is unlikely to have much effect since the reconstituted botulinum toxin will have a very short active life.
International Patent Application WO 0158472 describes a pharmaceutical composition comprising botulinum toxin and a polysaccharide. This application teaches that the polysaccharide stabilizes the neurotoxin. However, other studies, as discussed above, have shown that sacharrides are poor'stabilizers for botulinum toxin.
International Patent Application WO 04/060384 discloses a pharmaceutical botulinum toxin composition which includes a sequestration agent. The purpose of the sequestration agent is to prevent the diffusion of the botulinum toxin away from the site of injection. This does not address the need for stable compositions that can be applied to the surface of the skin.
There is no doubt that BOTOX® Cosmetic can smooth out fine lines and wrinkles for most users. However, there are several disadvantages associated with its use. The BOTOX® Cosmetic must be administered in a doctor's office. The injections can be painful and there may be bruising. Adverse side effects occur in some injection treated patients. Most common side effects for treatment of frown line include droopy eyelids, nausea, flu-like symptoms (fever etc.), headache and respiratory infections. Less frequent reactions may include facial pain, redness at the injection site, and muscle weakness at other sites. Repeated treatments may lead to permanent paralysis of facial muscles leaving the face expressionless.
Thus, there was a need for newer methods for stabilization of botulinum toxin. The present invention addresses that need. There was a further need for an alternative method of administering botulinum toxin that does not involve injections.